Evaluating the Use of TiO2 Nanoparticles for Toxicity Testing in Pulmonary A549 Cells

ORIGINAL RESEARCH Evaluating the Use of TiO 2 Nanoparticles for Toxicity Testing in Pulmonary A549 Cells Jana Bacova 1 , Petr Knotek 2 , Katerina Kopecka 2 , Ludek Hromadko 3 , Jan Capek 1 , Pavlina Nyvltova 1 , Lenka Bruckova 1 , Ladislava Schröterova 4 , Blanka Sestakova 4 , Jiri Palarcik 5 , Martin Motola 3 , Dana Cizkova 6 , Ales Bezrouk 7 , Jiri Handl 1 , Zdenek Fiala 8 , Emil Rudolf 4 , Zuzana Bilkova 1 , Jan M Macak 3,9 , Tomas Rousar 1 1 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic; 2 Department of General and Inorganic Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic; 3 Center of Materials and Nanotechnologies, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic; 4 Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic; 5 Institute of Environmental and Chemical Engineering, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic; 6 Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic; 7 Department of Medical Biophysics, Faculty of Medicine in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic; 8 Department of Preventive Medicine, Faculty of Medicine in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic; 9 Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic Correspondence: Tomas Rousar, Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic, Tel +420 466 037 707, Fax +420 466 036 361, Email T[email protected] Purpose: Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO 2 P25), are among the most produced nanomaterials worldwide. The broad use of TiO 2 P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO 2 P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO 2 P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO 2 P25 for biological testing in A549 cells and to evaluate their biological effects. Methods: We determined the size and crystallinity of TiO 2 P25. We used four techniques for TiO 2 P25 dispersion. We estimated the colloid stability of TiO 2 P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO 2 P25 (0–100 µg.mL −1 ) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. Results: We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO 2 P25. Under these conditions, TiO 2 P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO 2 P25 in A549 cells were met, similar results on the biological effects of TiO 2 P25 were obtained in two independent cell laboratories. Conclusion: We optimized the experimental conditions of TiO 2 P25 preparation for toxicity testing in A549 cells. The results presented here on TiO 2 P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO 2 P25 as a reference material. Keywords: titanium dioxide, nanoparticles, P25, nanotoxicity, A549 cells, dispersion Introduction A number of studies testing nanomaterial (NM) toxicity has been steadily increasing over past years. NMs can be used for various purposes based on their unique properties, which, in particular, are linked with their size below 100 nanometers in at least one of their dimensions. 1 However, the small size can also raise some questions on their biological effects in cells and organisms. Thus, studying biological effects of NMs, ie the estimation of their cytotoxicity or biocompatibility, is of great importance for our society. 2–5 International Journal of Nanomedicine 2022:17 4211–4225 4211 © 2022 Bacova et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). International Journal of Nanomedicine Dovepress open access to scientific and medical research Open Access Full Text Article Received: 19 May 2022 Accepted: 21 August 2022 Published: 13 September 2022 International Journal of Nanomedicine downloaded from https://www.dovepress.com/ on 13-Dec-2022 For personal use only. The lungs are one of the most common entrance points of NMs into the human body. 6 Several biological models have been introduced for testing of pulmonary toxicity. The most common approach to evaluate pulmonary toxicity is represented by in vitro models including human cell lines. 7–9 One of those, the human lung adenocarcinoma epithelial A549 cell line, was initiated in 1972. 10 A549 cells contain multilamellar cytoplasmic inclusion bodies typical of those found in type II alveolar epithelial cells. 10 A549 cells have been widely used in reports estimating pulmonary toxicity of NMs, eg silica (SiO 2 ), iron oxides (Fe x O y ), zinc oxide (ZnO), titanium dioxide (TiO 2 ), 11–13 silver (Ag) 14 nanoparticles and other specifically functionalized nanoparticles, 15 nanofibers, 16,17 nanosheets 18 and carbon-based nanomaterials. 19,20 A variety of TiO 2 NMs, eg nanoparticles, 21 nanofibers 17 and nanotubes, 22 has been developed. All these NMs have been evaluated for biological effects because TiO 2 has been used in medicine, material science and industry. In vitro studies describing cellular effects of TiO 2 nanoparticles (NPs) have been reported most frequently, especially using the pulmonary A549 cell line. 23 Interestingly, the reports on TiO 2 NPs biological effects have provided findings of different extent of NPs-induced toxicity in A549 cells. To date, more than 40 studies have reported results on the effects of 25 nm sized TiO 2 nanoparticles (P25) in A549 cells. Those studies have differed in dispersion techniques, tested concentration, incubation period, toxicity assay, or presence of fetal bovine serum. 24–27 The biological effect of TiO 2 P25 on proliferation and viability of A549 cells was estimated using cytotoxicity assays, including formazan-derived MTT 28 /XTT 29 /WST-1 30,31 and Trypan Blue Exclusion (TBE) tests. 24 An executive overview of these published results is provided in Table 1 that shows that the biological effect of TiO 2 P25 in A549 cells ranged from reporting negligible effects 32–36 to finding substantial cellular impairment. 11,37–40 Based on this discrepancy, we decided to estimate the factors influencing detected biological effects. Thus, the aim of the present study was to determine the optimal conditions for testing of biological effects of TiO 2 P25 in A549 cells, including material characterization, estimation of dispersion conditions, optimization of cell culture and toxicity testing. Then, we aimed to use the optimal parameters of TiO 2 P25 preparation for biological testing in two independent cellular laboratories and to compare obtained results. An essential topicality of present study can be also supported by frequent use of TiO 2 P25 as a comparative material for evaluation of biological effects in newly developed NMs. 21,37,41,42 Materials and Methods Chemicals and Materials Titanium dioxide nanoparticles (P25; anatase/rutile mixture, Product no. 718467, LOT MKCD 8503), glutaraldehyde, cacodylate buffer, osmium tetroxide, propylene oxide, lead citrate, Epon 812 and Durcupan, WST-1 reagent, monochlorobimane, formaldehyde, Triton X-100, phalloidin-FITC (phalloidin-fluorescein isothiocyanate) and fluorescence dye Hoechst 33258 were purchased from Sigma-Aldrich (USA). Uranyless was purchased from Delta Microscopies (France). Minimum Essential Medium, fetal bovine serum (FBS), pyruvate, glutamine, HEPES, penicillin, streptomycin, and Table 1 Overview of Reports Testing TiO 2 P25 Biological Effect in A549 Cells Detection of Cell Impairment [100 µg.mL −1 TiO 2 P25] Tested Dose of TiO 2 P25 [µg.mL −1 ] Sonication/ Duration Viability Test/ References No ≤ 800 Probe/10 min MTS 31 No ≤ 250 Probe/16 min WST-1 32 No ≤ 1000 N.D. WST-1 38 No ≤ 100 N.D. MTT 33 Yes ≤ 1000 Bath/30 min MTS 35 Yes ≤ 100 Probe/30 min MTT 36 Yes ≤ 200 Bath/15 min MTT 34 Yes ≤ 75 Bath/15 min MTT/WST-1 37 https://doi.org/10.2147/IJN.S374955 DovePress International Journal of Nanomedicine 2022:17 4212 Bacova et al Dovepress Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org) Dulbecco’s phosphate buffered saline (DPBS) were purchased from Invitrogen-Gibco (USA). Chloromethyl-2’,7’- dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA) was obtained from Thermo (USA). Multi-walled carbon nanotubes (JRCNM40003a, MWCNTs) were obtained from JRC Nanomaterials Repository as a reference material. Characterization of TiO 2 P25 The size and morphology of TiO 2 P25 were characterized by a field-emission scanning electron microscope JSM 7500F (SEM, JEOL, Japan). X-ray diffraction (XRD) analysis was carried out using Panalytical Empyrean with Cu tube and Pixcel 3D detector. The diffractometer was set up in Bragg–Brentano geometry. The diffractogram was taken at range 5– 80 degree 2Theta. Raman scattering spectrum of TiO 2 P25, excited by a laser operating at 785 nm, was obtained using a Dimension P2 (Lambda Solution, USA). 43 The topology of TiO 2 P25 surface was monitored on an atomic force microscope Dimension Icon (Bruker, Germany) in PeakForce Quantitative Nanoscale Mechanical mode using ScanAsyst-Air tips (k = 0.4 N/m) according to the described procedure. 44 TiO 2 P25 Dispersion Techniques TiO 2 P25 stock solutions (10 mg.mL −1 ) were prepared in distilled water. Different techniques were used to disperse TiO 2 P25, ie (1) manual shaking by hand in a tube, (2) sonication using ultrasonic probe UP400S, 400 W, 24 kHz (Hielscher Ultrasonics GmbH, Germany), equipped with titanium sonotrode H14 (14 mm in diameter) with the application of half of the cycle and maximal power, (3) FisherBrand FB15053H ultrasonic bath, 560 W (Fisher Scientific, UK), and (4) Ultraturrax® disperser T10 (IKA-Werke GmbH & Co. KG, Germany) equipped with a dispersion tool (S 10 D-7 G-KS-65) at 13,000 rpm. The dispersion of 100 µg.mL −1 TiO 2 P25 was carried out for up to 60 min. Then, the mean hydrodynamic diameter D H was measured by 90Plus/BI-MAS Analyzer (Brookhaven Instruments Corp., USA) using dynamic light scattering (DLS). D H values were measured for 30 s (n = 10) and these data were statistically processed according to ISO 13321/22412. 45 In addition, TiO 2 P25 stock solutions (10 mg.mL −1 ) were prepared in distilled water, 0.9% NaCl and Minimum Essential Medium for cell culture w/wo 10% FBS. The stock solutions were diluted to obtain the final concentration 100 µg.mL −1 TiO 2 P25, vortexed for 1 min and dispersed in ultrasonic bath K2, 60 W, 33 kHz (Kraintek, Slovakia) for 10 min. Average particle size was determined by dynamic light scattering using a Zetasizer Nano ZS (Malvern Panalytical Ltd., United Kingdom). The measurements were performed at 25 °C, with a scattering angle of 173°, using disposable sizing cuvettes. Each measurement was performed in 10 repeats after 30 s. Data were statistically processed. Endotoxin Contamination TiO 2 P25 were suspended in endotoxin-free water and diluted at 1 mg.mL −1 concentration. Nanoparticles were vigorously vortexed, sonicated for 15 min and centrifuged (15,000g; 15 min). The endotoxin concentration was measured in the supernatant using the PyroGene™ Recombinant Factor C Assay (Lonza, Blackley, UK). According to manufacturer´s instructions, the presence of endotoxin in a sample was calculated using the standard curve and results were expressed as endotoxin concentration in EU.mL −1 . Cell Culture The A549 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Minimum Essential Medium with 10% (v/v) FBS, 2 mmol.L −1 glutamine, 1 mmol.L −1 pyruvate, 10 mmol.L −1 HEPES, 50 μmol.L −1 penicillin/streptomycin and maintained at 37 °C in a sterile humidified atmosphere of 5% CO 2 . TiO 2 P25 treatment was initiated at 70% of confluence. The cells were proven to be Mycoplasma-free and the origin of the cells was confirmed by STR analysis. Cell Treatment with TiO 2 P25 For in vitro experiments, stock solutions of 10 mg.mL −1 TiO 2 P25 and MWCNTs were dispersed in culture medium w/wo FBS. Then, the stock solutions were vortexed for 1 min and sonicated for 10 min using ultrasonic bath K2. The working International Journal of Nanomedicine 2022:17 https://doi.org/10.2147/IJN.S374955 DovePress 4213 Dovepress Bacova et al Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org) solutions were prepared by dilution in the culture medium without phenol red to obtain final concentrations of 1, 10, and 100 μg.mL −1 . A549 cells were seeded into 96-well plates at density of 10×10 3 cells per well. After 24 h of seeding, the cells were exposed to 1, 10, and 100 μg.mL −1 TiO 2 P25. The cells were incubated with TiO 2 P25 at 37 °C in 5% CO 2 for 24 h. Unexposed cells were used as a negative control and MWCNTs were used as a positive control. For inter-laboratory comparison of TiO 2 P25-induced biological effects in A549 cells, the described above conditions for TiO 2 P25 preparation were used. Two independent laboratories, namely, Laboratory I (Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Czech Republic) and Laboratory II (Department of Medical Biology and Genetics, Charles University, Faculty of Medicine in Hradec Kralove, Czech Republic) prepared TiO 2 P25 stock solutions independently (10 mg.mL −1 TiO 2 P25 in cell culture medium with 10% FBS; sonication for 10 min using ultrasonic bath). Then, the working solutions were prepared by their dilution in the culture medium to obtain the final concentration 1–100 μg.mL –1 . A549 cells were seeded into 96-well plates at density of 10×10 3 cells per well for 24 h. Then, the cells were treated with TiO 2 P25 for 24 h and biological effect was tested using the WST-1 test. Dehydrogenase Activity Measurement The cell viability was assessed using the WST-1 test. The WST-1 test measures the activity of mitochondrial dehydrogenases. 46 After incubation with nanomaterials, 10 µL of WST-1 reagent was added to each well containing cells in 100 µL of culture medium according to the manufacturer’s instructions. After 1 h, the change of absorbance was measured at the wavelength of 440 nm using SPARK microplate reader (Tecan, Austria) or at 450 nm with 650 nm reference wavelength using SPEKTRAFluor Plus (Tecan, Austria) while incubated at 37 °C. The dehydrogenase activity was expressed as the percentage of total cellular dehydrogenases activity relative to that in control cells (control = 100%). Measurement of Glutathione Levels The glutathione (GSH) levels were measured using an optimized monochlorobimane assay. 47 The working solution of monochlorobimane (MCB) was prepared fresh at the time of analysis by dilution in Dulbecco’s phosphate buffer and tempered at 37 °C. After the treatment, 20 μL of the MCB solution was added to the cells in 96-well plates and the measurement started immediately. The final concentration of MCB in a well was 40 μmol.L –1 . The fluorescence intensity (Ex/Em = 394/490 nm) was measured kinetically for 20 min using SPARK microplate reader (Tecan, Austria). The fluorescence was expressed as the slope of a fluorescence change over time. GSH levels were expressed as the percentage relative to GSH levels in control cells (control = 100%). Transmission Electron Microscopy A549 cells were fixed in 3% glutaraldehyde (in 0.1 mol.L −1 cacodylate buffer, pH 7.2) after quick and gentle wash in 0.1 mol.L −1 cacodylate buffer (pH 7.2) directly in the culture flask, for 5 min at 37 °C and then for 3 h at room temperature. Following rinsing in 0.1 mol.L −1 cacodylate buffer (pH 7.2), the cells were post-fixed in 1% osmium tetroxide (in 0.1 mol.L −1 cacodylate buffer, pH 7.2) for 1 h at room temperature, washed in cacodylate buffer (0.1 mol.L −1 , pH 7.2) and dehydrated in graded alcohols (50%, 75%, 96% and 100%). For clarification, propylene oxide was used and subsequently, the cells were embedded in the mixture of Epon 812 and Durcupan (polymerization for 3 days at 60 °C). Ultrathin sections cut on Ultrotome Nova (LKB, Broma, Sweden) were collected onto formvar carbon-coated copper grids (Plano, Wetzlar, Germany) and counterstained with uranyl acetate using Uranyless and lead citrate. In a JEOL JEM-1400Plus transmission electron microscope (TEM, at 120 kV; JEOL, Japan) the ultrathin sections were observed, and images were captured with the integrated 8Mpix CCD camera and using software TEM Center (ver. 1.7.1537; JEOL). Detection of Nuclear Condensation and Fragmentation To measure nuclear condensation and fragmentation in intact cells, we used a fluorescence dye: Hoechst 33258 (H33258). 48 After treatment with tested nanomaterials for 24 h, the cells grown in a 96-well plate were centrifuged (5 min; 8,000g; RT). Then, 70 µL of a supernatant was replaced with 70 µL of phosphate-buffered saline and 10 µL of H33258 solution was added to a well. The final concentrations of H33258 in a well was 2 µg.mL −1 . Then, the cells were incubated with H33258 for 5 min https://doi.org/10.2147/IJN.S374955 DovePress International Journal of Nanomedicine 2022:17 4214 Bacova et al Dovepress Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org) and spectrofluorometric measurement was performed at Ex/Em = 352/461 nm using SPARK microplate reader (Tecan, Austria) while incubated at 37 °C. The samples were measured at least in triplicates. After background subtraction, the fluorescence signal was presented in Relative Fluorescence Units (RFU) as mean ± SEM. Detection of Reactive Oxygen Species (ROS) Chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA) was used as an intracellular probe to detect ROS production. The working solution was prepared fresh at the time of analysis by dilution in culture medium. After 24 h incubation with TiO 2 P25 and MWCNTs, 67.5 µL of CM-H 2 DCFDA was added to cells to be loaded for 90 min. The final concentration of CM-H 2 DCFDA in a well was 5 µmol.L −1 . Then, the cells were washed with phosphate buffered saline and the fluorescence (Ex/Em = 485/535 nm) was measured for 60 min using SPARK microplate reader (Tecan, Austria). The ROS levels were expressed as the percentage relative to ROS levels in control cells (= 100%). Fluorescence Microscopy To stain actin filaments, we used a phalloidin-FITC dye. A549 cells were seeded at density of 10×10 3 cells/well of a chamber slide. After 24 h, the cells were exposed to 1, 10 and 100 µg.mL −1 TiO 2 P25 or 100 µg.mL −1 MWCNTs. After the treatment, the A549 cells were fixed by 3.7% formaldehyde (5 min; 37 °C; dark) and permeabilized by 0.1% Triton X-100 (15 min; 37 °C; dark). Then, 100 μL of phalloidin-FITC (1 μmol.L −1 ) was incubated for 40 min at 37 °C. After dye loading, the cells were washed two times with phosphate-buffered saline. The actin filaments (FITC filter, 480/30 nm) and morphology of A549 cells using phase contrast were observed with a fluorescence microscope Eclipse 80i (Nikon, Japan). Data Evaluation and Statistical Analysis All experiments were repeated at least three times independently. Three replicates were used in each independent experiment. In addition, we evaluated the interference of tested nanomaterials with the assays. We found no significant interference with most of the used assays when the background was below 10% of that in negative controls. Only in nuclear condensation and fragmentation H33258 assay, did a larger extent of interference occur in 100 µg.mL −1 TiO 2 P25 and 100 µg.mL −1 MWCNTs treated cells. The results are expressed as mean ± SD. The analysis of variance followed by Bonferroni post-test was used to perform the mean comparison at significance level p = 0.05. Results Characterization of TiO 2 P25 Firstly, we characterized shape and size of commercially available TiO 2 P25 nanoparticles (TiO 2 P25) using scanning electron microscopy. We found that TiO 2 P25 were supplied in a form of agglomerates with an average size over 1 µm in maximal dimension (Figure 1A). Those agglomerates were composed of primary particles with size 30±10 nm (Figure 1B) but the primary particles were obviously interconnected by solid bridges to each other. Figure 1 SEM images of TiO 2 P25 nanoparticles at two different magnifications. International Journal of Nanomedicine 2022:17 https://doi.org/10.2147/IJN.S374955 DovePress 4215 Dovepress Bacova et al Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org) The phase composition of TiO 2 P25 was analyzed by X-ray diffraction analysis (Figure 2). The sample of TiO 2 P25 consisted of 90.4% (wt) of anatase (ICDD:00–021-1272) and 9.6% (wt) of rutile (ICDD:00–021-1276) phases. The crystallite size for anatase phase of ≈ 27 nm was determined by Rietveld method. These results are in in accordance with manufacturer´s information. Dispersion of TiO 2 P25 A proper dispersion of TiO 2 P25, ie a separation of aggregates into ideally a mixture of well-separated single particles, is a crucial point for assessment of their biological effects. Thus, the rate of dispersion of TiO 2 P25 in distilled water was tested using four techniques. Out of these, three techniques were chosen according to the most frequently used procedures in the literature, namely: hand shaking, ultrasonication in a bath and by an arc probe. In addition, we used Ultra-turrax instrument ensuring TiO 2 P25 dispersion using the maximal shear forces. In addition to the hydrodynamic diameter measurement of TiO 2 P25 dispersed for 40 min (Figure 3A), we tested the effect on duration of dispersion (Figure 3B). Our results showed that the maximal rate of dispersion of TiO 2 P25 in distilled water was found in nanoparticles dispersed using ultrasonic probe and bath. After the dispersion using ultrasonic probe, we detected a fraction of TiO 2 P25 sized under 100 nm (Figure 3A). In TiO 2 P25 dispersed using ultrasonic bath, the smallest fraction of nanoparticles was about 150 nm in size. The use of both techniques, however, provided also fractions of Figure 2 XRD patterns of TiO 2 P25 nanoparticles showing anatase (black) and rutile (red) peaks. The sample of TiO 2 P25 consisted of 90.4% (wt) of anatase (ICDD:00–021-1272) and 9.6% (wt) of rutile (ICDD:00–021-1276) phases. Figure 3 Results of dispersion of TiO 2 P25 in distilled water (100 µg.mL −1 ) using four dispersion techniques: hand shaking, ultrasonic (= US) bath, US probe and Ultraturrax. The particle size distribution was measured - (A) directly after finished 40 min dispersion, or (B) after various dispersion times between 5–60 min. https://doi.org/10.2147/IJN.S374955 DovePress International Journal of Nanomedicine 2022:17 4216 Bacova et al Dovepress Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org) dispersed nanoparticles being larger than 200 nm (Figure 3A). The use of Ultra-turrax and hand shaking caused dispersion of TiO 2 P25 at limited extent only. Our results on the extent of TiO 2 P25 dispersion in time (Figure 3B) showed that the effective hydrodynamic diameter remained predominantly stable. The duration to obtain the maximal rate of dispersion was determined to be at least 10 min. The results from the testing of different dispersion techniques were obtained in TiO 2 P25 diluted in distilled water. Generally, the testing of NMs in cells, however, requires a use of culture media containing all necessary ingredients to ensure the cell growth and proliferation. Thus, we tested the behavior of TiO 2 P25 in distilled water in comparison to the dispersion stability in saline solution and Minimum Essential cell culture medium with or without addition of 10% fetal bovine serum. Our previous results showed that the dispersion of TiO 2 P25 using ultrasonic probe or ultrasonic bath provided comparable results on dispersion of nanoparticles in complex matrix of cell culture medium. Thus, we used the dispersion of TiO 2 P25 nanoparticles using ultrasonic bath for 10 min following the procedure described below. We determined the relation of obtained extent of TiO 2 P25 dispersion and used environment (Figure 4). As expected, the dispersion of TiO 2 P25 in distilled water provided a small fraction of TiO 2 P25 about 150 nm in size but the large population of nanoparticles aggregates remained over 400 nm in size. No dispersed TiO 2 P25 aggregates under 400 nm in size were detected in NaCl solution and in culture medium without FBS. On the other hand, the presence of FBS in cell culture medium stabilized the dispersion of nanoparticles providing the size of TiO 2 P25 aggregates at about 100 nm in 10% of nanoparticles. The final evaluation of our data on dispersion of TiO 2 P25 provided essential information on necessity of FBS presence in assessment of biological effects of TiO 2 P25 in cell culture media to ensure as large as possible dispersion. We conclude that a number of factors have been influencing the accomplishment of proper TiO 2 P25 dispersion, ie dispersion technique, duration, ingredients in the cell culture medium and also the interval between preparation of TiO 2 P25 suspension and addition to cultured cells. Thus, in all following experiments, we prepared TiO 2 P25 suspensions using sonication in ultrasonic bath (10 min) in cell culture medium with FBS ensuring the colloidal stability. Effect of FBS in TiO 2 P25 Treatment of A549 Cells According to the frequent use of A549 cells in the literature, 12,28,49,50 we used this cellular model for testing of TiO 2 P25 biological effects too. Firstly, we tested TiO 2 P25 for potential endotoxin contamination. We found that the concentration of endotoxin in the sample occurred under the detection limit of the assay (˂0.005 EU.mL −1 ). Thus, tested TiO 2 P25 were proven to be endotoxin-free. Then, we estimated the influence of the fetal bovine serum presence on TiO 2 P25-induced biological effects. A549 cells were treated with 0–100 µg.mL −1 TiO 2 P25 w/wo FBS. After 24 h, we estimated an effect of TiO 2 P25 on the cell viability Figure 4 Results of TiO 2 P25 size distribution (100 µg.mL −1 ) in different solutions: distilled water, 0.9% NaCl, cell culture medium w/wo 10% fetal bovine serum (FBS) for 10 min using ultrasonic bath. Data are presented as intensity distribution (A) and cumulative intensity (B) of TiO 2 P25 size. International Journal of Nanomedicine 2022:17 https://doi.org/10.2147/IJN.S374955 DovePress 4217 Dovepress Bacova et al Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org) and glutathione levels measured using the WST-1 test and monochlorobimane, respectively. The results are presented in Table 2. In A549 cells incubated with or without FBS, we found that none of the tested TiO 2 P25 concentrations affected cellular dehydrogenase activity significantly in comparison to untreated cells. A slight decrease of dehydrogenase activity to 97 ± 4% was detected only in 100 µg.mL −1 TiO 2 P25 treated A549 cells with FBS. Measurement of glutathione levels, as an essential intracellular antioxidant, detected a significant glutathione depletion in 100 µg.mL −1 TiO 2 P25 treated A549 cells grown both with and without FBS. The results are presented in Table 2. In addition, mild, but significant glutathione depletion was observed in A549 cells with FBS exposed to 10 µg.mL −1 TiO 2 P25. Transmission electron microscopic analyses were carried out to evaluate the effect of 10% FBS on TiO 2 P25 cellular acting. TEM photomicrographs (Figure 5) showed internalization of TiO 2 P25 (100 µg.mL −1 ) after 24 h of incubation of A549 cells both in presence and absence of FBS. TiO 2 P25 occurred in the cytoplasm predominantly in simple or double-membrane vesicles. Near the cell surface, many slender cytoplasmic projections were formed and TiO 2 P25 were accumulated in their vicinity. The most obvious difference between A549 cells incubated with TiO 2 P25 w/wo FBS was that the nanoparticles dispersed in FBS-free medium aggregated and accumulated more around the cells (Figure 5A) comparing the cells incubated in FBS containing medium (Figure 5B). In accordance with presented results, we concluded that presence of FBS in cell culture medium ensures proper TiO 2 P25 dispersion necessary for valuable estimation of TiO 2 P25 effect in cells. Thus, we used TiO 2 P25 treatment of A549 cells in presence of 10% FBS in all following experiments. Table 2 TiO 2 P25 Cytotoxicity Evaluation in A549 Cells. Dehydrogenase Activity (= Cell Viability, WST-1 Test) and Glutathione Levels Were Assayed in A549 Cells Treated with 0–100 µg.mL −1 TiO 2 P25 in Culture Medium w/wo Fetal Bovine Serum (FBS) for 24 h. The Results are Expressed as Mean ± SD (p < 0.001, Compared to Untreated Cells; Three Independent Experiments) Cell Culture TiO 2 P25 [µg.mL −1 ] Dehydrogenase Activity Glutathione Level Without FBS 0 100 ± 3% 100 ± 3% 1 99 ± 5% 100 ± 5% 10 101 ± 4% 98 ± 5% 100 97 ± 4% 85 ± 7% (p ˂ 0.001) With FBS 0 100 ± 3% 100 ± 4% 1 97 ± 5% 104 ± 6% 10 98 ± 8% 91 ± 4% (p ˂ 0.001) 100 97 ± 9% 87 ± 4% (p ˂ 0.001) Figure 5 TEM images of A549 cells treated with TiO 2 P25 w/wo fetal bovine serum. (A), without FBS; (B), with FBS. The pictures confirmed internalization of TiO 2 P25 in the cytoplasm in vesicles (arrowheads, inserts). TiO 2 P25 accumulated in the vicinity of slender cytoplasmic projections (arrows). Scale bar = 5 µm (mag. 2500x); insert (A) (mag. 11,100x); insert (B) (mag. 14,500x). https://doi.org/10.2147/IJN.S374955 DovePress International Journal of Nanomedicine 2022:17 4218 Bacova et al Dovepress Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org) Estimation of TiO 2 P25 Effect in A549 Cells In addition to WST-1 and glutathione tests, we used three additional methods for characterizing the TiO 2 P25 effects in A549 cells in more detail, ie determination of nuclear condensation and fragmentation, ROS production and assessment of cell morphology. To evaluate the cellular effect, we tested TiO 2 P25 (0–100 µg.mL −1 ) in comparison to MWCNTs (100 µg.mL −1 ) used as a positive control (Figure 6A). After 24 h, we found that none of tested TiO 2 P25 concentrations induced significant nuclear condensation and fragmentation in comparison to untreated cells. Only a mild increase of DNA condensation was found with 100 µg.mL −1 TiO 2 P25, implying that TiO 2 P25 treatment did not cause an induction of apoptotic cell death. On the other hand, a significant increase of nuclear condensation and fragmentation was detected in MWCNTs treated A549 cells. To observe any induction of an oxidative stress after TiO 2 P25 treatment in A549 cells, we investigated the production of reactive oxygen species using a spectrofluorometric probe detecting entire ROS production. After 24 h of incubation, we did not observe any significant induction of ROS production in tested TiO 2 P25 in comparison to untreated A549 cells (Figure 6B). MWCNTs treatment, however, induced significant increase of ROS production. Fluorescence staining of actin filaments and phase contrast microscopy were used for a visual evaluation of TiO 2 P25treated A549 cells (Figure 6C). Typical epithelial morphology was found in both untreated and TiO 2 P25-treated A549 cells, showing no significant effect of TiO 2 P25 treatment. On the other hand, incubation of A549 cells with MWCNTs caused changes found in photomicrographs, lowering the number of cells and changing their morphology. Finally, Figure 6 Estimation of TiO 2 P25 effects in A549 cells. A549 cells were treated with TiO 2 P25 (0–100 µg.mL −1 ) and MWCNTs (100 µg.mL −1 ) with FBS for 24 h. (A) nuclear condensation and fragmentation, (B) ROS production and (C) A549 cells morphology (scale bar = 10 µm) were estimated. The results are expressed as mean ± SD (***p < 0.001, compared to untreated cells). International Journal of Nanomedicine 2022:17 https://doi.org/10.2147/IJN.S374955 DovePress 4219 Dovepress Bacova et al Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org)